Prenatal diagnosis is currently performed using conventional cytogenetic analysis (such as karyotyping) or DNA analysis (such as QF-PCR), which require fetal genetic material to be obtained by amniocentesis, chorionic villus sampling or chordocentesis. However, these are invasive procedures and are associated with a significant risk of fetal loss (0.5 to 1% for chorionic villus sampling and amniocentesis) (Hultén, M. A. et al. (2003) Reproduction 126:279-297). The need for effective prenatal diagnostic tests is particularly acute in the case of Down Syndrome, also known as trisomy 21 syndrome, which is considered to be the most frequent form of mental retardation, with an incidence of 1 in 700 child births in all populations worldwide. However, due to the current risk of prenatal testing, prenatal diagnosis is only offered to high risk pregnancies (6-8% of all pregnancies), which are assessed based on maternal serum screening and fetal ultrasonography programs. Thus, there is an urgent need for the development of diagnostic procedures that do not put the fetus at risk, which is commonly termed as noninvasive prenatal diagnosis.
Free fetal DNA (ffDNA) has been discovered in the maternal circulation during pregnancy (Lo, Y. M. et al. (1997) Lancet 350:485-487), and has become a focus for alternative approaches toward the development of noninvasive prenatal tests. ffDNA has been successfully used for the determination of fetal sex and fetal RhD status in maternal plasma (Lo, Y. M. et al. (1998) N. Engl. J. Med. 339:1734-1738; Bianchi, D. W. et al. (2005) Obstet. Gynecol. 106:841-844). Nevertheless, direct analysis of the limited amount of ffDNA (3 to 6%) in the presence of excess of maternal DNA is a great challenge for the development of noninvasive testing for fetal aneuploidies.
Recent advances in the field have shown that physical and molecular characteristics of the ffDNA can be used for its discrimination from circulating maternal DNA or as a means of fetal DNA enrichment (Chan, K. C. et al. (2004) Clin. Chem. 50:88-92; Poon, L. L. et al. (2002) Clin. Chem. 48:35-41). For example, size fractionation has been used on plasma DNA to enrich for fetal DNA because fetal DNA is generally shorter in length than maternal DNA in the circulation (Chan, K. C. et al. (2004), supra). Furthermore, based on evidence that ffDNA in maternal plasma is of placental origin, epigenetic differences between maternal peripheral (whole) blood and placental DNA have been used to detect a hypomethylated gene sequence (maspin/SERPINB5) in maternal plasma derived from the fetus (Masuzaki, H. et al. (2004) J. Med. Genet. 41:289-292; Fiori, E. et al. (2004) Hum. Reprod. 19:723-724; Chim, S. S. et al. (2005) Proc. Natl. Acad. Sci. USA 102:14753-14758). Subsequently, a small number of additional differential fetal epigenetic molecular markers have been described, including the RASSF1A gene on chromosome 3, as well as a marker on chromosome 21 (Chiu, R. W. et al. (2007) Am. J. Pathol. 170:941-950; Old, R. W. et al. (2007) Reprod. Biomed. Online 15:227-235; Chim, S. S. et al. (2008) Clin. Chem. 54:500-511).
Although these studies have demonstrated that epigenetic differences between fetal DNA (placental DNA obtained from chorionic villus sampling) and maternal peripheral blood DNA may serve as potential fetal molecular markers for noninvasive prenatal diagnosis, only a limited number of genomic regions have been identified or tested to date. A number of studies have focused on single gene promoter regions (Chim, S. S. et al. (2005) supra; Chiu, R. W. et al. (2007, supra), whereas others have investigated CpG islands on chromosome 21 (Old, R. W. et al. (2007) supra; Chim, S. S. et al. (2008) supra), which however cover only a small fraction of the chromosome (Fazzari, M. J. et al. (2004) Nat. Rev. Genet. 5:446-455).
Current methods developed using ffDNA for noninvasive prenatal diagnosis are subject to a number of limitations. One method being investigated involves the use of methylation-sensitive restriction enzymes to remove hypomethylated maternal DNA thus allowing direct polymerase chain reaction (PCR) analysis of ffDNA (Old, R. W. et al. (2007), supra). However, the requirement for regions of differentially methylated DNA to contain a restriction site for recognition by methylation-sensitive restriction enzymes limits the number of regions suitable for testing. Another method being investigated involves the use of sodium bisulfite conversion to allow the discrimination of differential methylation between maternal and fetal DNA. In this approach, sodium bisulfite conversion is followed by either methylation-specific PCR or methylation sensitive single nucleotide primer extension and/or bisulfite sequencing (Chim, S. S. et al. (2005) supra; Chiu, R. W. et al. (2007) supra; Chim, S. S. et al. (2008) supra). This approach, however, has two main problems. Firstly, the accurate analysis of the methylation status after bisulfite conversion depends on the complete conversion of unmethylated cytosines to uracils, a condition rarely achieved. Secondly, the degradation of DNA obtained after bisulfite treatment (described in Grunau, C. et al. (2001) Nucl. Acids Res. 29:E65-5) complicates even further the testing and quantification of extremely low amounts of fetal DNA.
Another recent approach has been to directly sequence cell-free DNA from the plasma of pregnant women, using a high throughput shotgun sequencing technique (Fan, H. C. et. al (2008) Proc. Natl. Acad. Sci. USA 105:16266-71; Chiu, R. W. et. al. (2008) Proc. Natl. Acad. Sci USA. 105:20458-63). However, this approach is technologically demanding and the high cost of this approach makes its application extremely difficult to the majority of diagnostic laboratories.
Accordingly, additional approaches and methods for noninvasive prenatal diagnosis of fetal aneuploidies are needed, to reduce the risk of fetal loss and to allow for screening of all pregnancies, not just high risk pregnancies.